S Mimura1, T Masuda, T Matsui, H Takisawa. 1. Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
Abstract
BACKGROUND: In eukaryotes, chromosomal DNA is licensed to be replicated through the sequential loading of the origin recognition complex, Cdc6 and mini-chromosome maintenance protein complex (MCM) onto chromatin. However, how the replication machinery is assembled onto the licensed chromatin during initiation of replication is poorly understood. RESULTS: Using Xenopus egg extracts, we have investigated the role of Cdc45 in the loading of various replication proteins onto chromatin at the onset of S phase, and found that Cdc45, which required MCM for its loading, was essential for the sequential loading of replication protein A (RPA), DNA polymerase alpha and proliferating cell nuclear antigen (PCNA) onto chromatin. The assembly of DNA polymerase epsilon onto chromatin required Cdc45 but did not require DNA polymerase alpha. Analysis of nuclease-digested chromatin fractions shows that Cdc45 formed a stable complex with either MCM or DNA polymerase alpha on chromatin. CONCLUSIONS: These results demonstrate a central role for Cdc45 in activation of the licensed chromatin to form replication complexes at the onset of S phase, and suggest that Cdc45 has a dual role in the initiation of DNA replication: the unwinding of DNA and the recruiting of DNA polymerases onto DNA.
BACKGROUND: In eukaryotes, chromosomal DNA is licensed to be replicated through the sequential loading of the origin recognition complex, Cdc6 and mini-chromosome maintenance protein complex (MCM) onto chromatin. However, how the replication machinery is assembled onto the licensed chromatin during initiation of replication is poorly understood. RESULTS: Using Xenopus egg extracts, we have investigated the role of Cdc45 in the loading of various replication proteins onto chromatin at the onset of S phase, and found that Cdc45, which required MCM for its loading, was essential for the sequential loading of replication protein A (RPA), DNA polymerase alpha and proliferating cell nuclear antigen (PCNA) onto chromatin. The assembly of DNA polymerase epsilon onto chromatin required Cdc45 but did not require DNA polymerase alpha. Analysis of nuclease-digested chromatin fractions shows that Cdc45 formed a stable complex with either MCM or DNA polymerase alpha on chromatin. CONCLUSIONS: These results demonstrate a central role for Cdc45 in activation of the licensed chromatin to form replication complexes at the onset of S phase, and suggest that Cdc45 has a dual role in the initiation of DNA replication: the unwinding of DNA and the recruiting of DNA polymerases onto DNA.
Authors: Juraj Gregan; Karola Lindner; Lydia Brimage; Roger Franklin; Mandana Namdar; Elizabeth A Hart; Stephen J Aves; Stephen E Kearsey Journal: Mol Biol Cell Date: 2003-06-13 Impact factor: 4.138